Compositions and methods for treating bone diseases

ABSTRACT

Certain aspects of the present invention generally relate to compositions and methods for treating a bone disease, for example a genetic bone disease. In one embodiment the bone disease is fibular hemimelia, proximal femoral focal deficiency, tarsal coalition or humeroradial synostosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present patent application claims the benefit of the filing date ofU.S. Provisional Patent Application No. 62/215,987, filed Sep. 9, 2015,the contents of which is hereby incorporated by reference.

TECHNICAL FIELD

The present invention generally relates to compositions and methods fortreating a bone disease, for example a genetic bone disease. In oneembodiment, the bone disease is fibular hemimelia, proximal femoralfocal deficiency, tarsal coalition or humeroradial synostosis.

BACKGROUND

Fibular hemimelia (FH) is a disease of congenital limb deficiencycharacterized by the partial or total absence of the fibula. Itrepresents the most common deficiency of long bones. The cause of FHdisease remains unknown. It was first described by Gollier in 1698(Coventry and Johnson, 1952). It has been estimated that there isapproximately 1 in 50,000 cases live births (Florio et al., 1999). FHexhibits as a clinical spectrum from partial absence to complete absenceof the fibular bone (Achterman and Kalamchi, 1979). The appearance ofthe developmental anomaly is not only absence of the fibula but also asfollows: the femur is short, the tibia is bowed, the foot is deformed inan equinovalgus position, and presents tarsal coalition. Absence oflateral rays of the foot may be seen in patients with severe FH disease.

Proximal femoral focal deficiency (PFFD) is a rare congenital anomalycharacterized by abnormal development of the proximal femur (Gillespieet al., 1983). Symptoms range from an absence of the entire femur andabnormal development of the pelvis to a hypoplastic femur of normalconfiguration. The disorder is unilateral in 90% of the cases. It iscommonly linked with the absence or shortening of leg bone and theabsence of a kneecap. The lower extremity is abducted, flexed andexternally rotated through the upper femur. Other features of PFFDinclude the dislocation or instability of the joint between the femurand the kneecap, a shortened tibia or fibula, and foot deformities.

Tarsal coalition (TC) is a fibrous, cartilaginous, or bony connection oftwo or more tarsal bones. Foot pain is the typical symptom. Althoughusually developing during adolescence, the onset of aching pain in thefoot tends to correlate with age at which the coalition bar ossifies(Kulik et al., 1996). Classic presenting symptoms are that the peronealspastic flat foot, with the hindfood held in eversion and spasm of theperoneals noted on attempts to invert the foot. The most common tarsalcoalitions involve the calcaneonavicular and talocalcaneal joints.

Humeroradial synostosis (HRS) is rare congenital abnormality in whichthere is fusion between humerus and radius. HRS is categorized bydeformity. Class I is associated with ulnar ray hypoplasia and elbowextension. Class II synostoses are not associated with hypolasia wherethe elbow is flexed (McIntyre et al., 2002). HRS often causes littlefunctional disability. Both sporadic and genetic cases of humeroradialsynostosis are encountered.

The etiology of fibular hemimelia (FH), proximal femoral focaldeficiency (PFFD), tarsal coalition (TC) and humeroradial synostosis(HRS) disease are unclear, but genetic and environmental factors aregenerally acknowledged. Understanding the pathological mechanisms ofthese diseases will help the prevention and treatment of the diseases.

SUMMARY OF THE PREFERRED EMBODIMENTS

One aspect of the present invention provides a method for treating agenetic bone disease present in a subject. In one embodiment, the methodincludes administering to a subject in need of such treatment acomposition comprising a therapeutically effective amount of aninhibitor of beta-catenin signaling. The disease may be, for example,fibular hemimelia (FH), proximal femoral focal deficiency (PFFD), tarsalcoalition (TC) or humeroradial synostosis (HRS).

The composition may be administered in-utero and/or after birth of thesubject. For example, the composition may be administered in the thirdtrimester of pregnancy. The subject may be a subject having an elevatedlevel of beta-catenin signaling. In certain embodiments, the inhibitorof beta-catenin signaling is LGK-974, IWP-2, iCRT3, iCRT 14, ICG 001,XAV-939, KY02111, or a combination of at least two of these compounds.

Another aspect of the invention provides a method for treating anorthopedic disease, including administering to a subject in need of suchtreatment a composition including a therapeutically effective amount ofan inhibitor of bone morphogenetic protein (BMP) signaling. The diseasemay be, for example, fibular hemimelia (FH), proximal femoral focaldeficiency (PFFD), tarsal coalition (TC) or humeroradial synostosis(HRS).

The composition may be administered in-utero and/or after birth of thesubject. For example, the composition may be administered in the thirdtrimester of pregnancy. The subject may be a subject having an elevatedlevel of BMP signaling. In certain embodiments, the inhibitor of BMPsignaling dorsomorphin dihydrochloride, K 02288, DMH-1, LDN 193189trihydrochloride or a combination of at least two of these compounds.

Another aspect of the invention provides a method for identifyingcompounds for treating a genetic orthopedic disease. In one embodiment,the method includes administering a test compound to an Axin1^(Prx1)mouse embryo and examining an elbow joint of the embryo to determine theextent of separation of humerus, radius and ulna. Compounds that exhibitat least a predetermined separation of the humerus, radius and ulna areidentified as compounds for treating the genetic orthopedic disease. Inone embodiment, the examination is by x-ray analysis.

BRIEF DESCRIPT ON OF THE DRAWINGS

FIG. 1 shows defects in fibular development were observed inAxin1^(Prx1) conditional KO embryos and postnatal mice. Alizerinred/Alcian blue staining was performed using hind limbs of E13.5 and16.5 embryos and P7 postnatal mice. Axin1^(Prx1) KO embryos and miceshowed partial development of fibula. Cre- embryos (controls) showedmineralization of fibula at E16.5 or P7 stages. In contrast, thepartially developed fibula of Axin1^(Prx1) KO embryo and mouse did notmineralize even at day 7 of postnatal stage.

FIG. 2(a) shows defects in fibular development were observed inAxin1^(Prx1) conditional KO mice. 52 Axin1^(Prx1) KO mice were generatedand all of them showed defects in fibular development. Radiographicanalysis showed that the absence of the fibula in some of Axin1^(Prx1)mice was complete (1-week-old, lower panel) or almost complete (>50%loss, 27/52) where only a distal, vestigial fragment was present(5-week-old, tower panels). The other Axin1^(Prx1) mice had partialabsence of the fibula (30-50% loss, 23/52) (4-week-old, lower panel) inwhich the proximal portion of the fibula was absent while the distalportion was present but could not support the ankle.

FIG. 2(b) shows defects in fibular development and increased bonedensity were observed Axin1^(Prx1) KO Defect mice. X-ray radiographicanalysis showed that partial developed but mineralized fibulae (mildfibular hypoplasia) were observed in two of 8-week-old Axin1^(Prx1) KOmice (<30% loss, 2/52) (2nd left and far right) (upper arrows). Inaddition, bone mineral density was also increased in Axin1^(Prx1) KOmice (lower arrows).

FIG. 2(c) shows defects in fibular development in Axin1^(Prx1) KO mice.Fibular tissues were dissected from 8-week-old Cre-negative andAxin1^(Prx1) KO mice. Histological analysis (Alcian blue/H&E staining,lower panel) showed that disorganized fibular structure, abnormalcartilage development and significant increase in bone mass was observedin Axin1^(Prx1) KO mice.

FIG. 3 shows normal fibular development observed in Axin1^(Osx) andAxin1^(Col2) KO mice. To determine if deletion of Axin1 in osteoblastprecursor cells or in chondrocytes will exhibit similar defects infibular development, we generated Axin1^(Osx1) and Axin1^(Col2)conditional KO mice. Radiographic analysis showed that fibulardevelopment was normal in both Axin1^(Osx1) and Axin1^(Col2) KO mice(4-week-old).

FIG. 4 shows the fusion of elbow joint observed in Axin1^(Prx1) KOembryos and postnatal mice. Alizerin red/Alcian blue staining wasperformed in forelimbs of E13.5 and E16.5 embryos and postnatal day 7mice. Red (upper) arrows indicate fusion of humerus and radius at E13.5,E16.5 and P7 Axin1^(Prx1) KO embryos and mice. Black (lower) arrowindicates lateral fusion of humerus and ulna at E16.5 Axin1^(Prx1) KOembryos.

FIG. 5(A) shows that Axin1^(Prx1) KO mice have joint fusion defects inforelimb. X-ray radiographic analysis (P12 mice) showed humeroradial andhumerolunar fusions in Axin1^(Prx1) KO mice (red arrows, A: lowerpanel). FIG. 5(B) μCT analysis (P30 mice of three dimensional volumetricreconstructions of elbow joint and medial cross sections through thereconstructed volumes were generated. Red (left) arrows indicate fusionof humerus (H) and radius (R). Green (right) arrow indicates fusion ofhumerus (H) and ulna (U). It is worth noting that fusion between humerusand radius is a key characteristic of humeroradial synostosis in human.

FIG. 6, Axin1^(Prx1) KO mice have joint fusion defects in forelimb.Histological analysis of the elbow joint of Cre⁻and Axin1^(Prx1) KOmice. Alcian Blue/Hematoxylin & Orange G stained sections of elbow ofE16.5 embryos and and postnatal mice. The humerus H), radius and ulnaare labeled. Axin1^(Prx1) KO mice show fusion of humerus and radius(black (upper) arrows) and fusion of humerus and ulna (green (lower)arrow).

FIG. 7(A-B) shows fusion of tarsal elements observed in Axin1^(Prx1) KOmice. Fusion of 1st, 2nd, 3rd, and 4th/5th tarsal elements and centralein hind limb was observed by X-ray radiographic (A) and μCT analyses (B)in P15 Axin1^(Prx1) KO mice (arrows). This is tarsal coalition (TC) likephenotype, (1) 1st distal tarsal, (2) 2nd distal tarsal, (3) 3rd distaltarsal, (4/5) 4th/5th distal tarsal, (C) Centrale, (T) Tibiale, (I)Intermedium (astragalus), (F) Fibulare (calcaneum)

FIG. 8 shows Axin1^(Prx1) KO mice having defects in deltoid tuberosityin forelimb. μCT analysis of forelimb of 3- and 6-week-old Cre- andAxin1^(Prx1) mice. 3D images of forelimb were generated. The deltoidtuberosity in Axin1^(Prx1) KO mice is much smaller than that of Cre-mice. Arrows indicate defects of deltoid tuberosity at the forelimb ofAxin1^(Prx1) KO mice.

FIG. 9 shows Axin1^(Prx1)/Axin2^(+/−)double mutant mice having moresevere defects in skeletal development. Axin1^(Prx1)/Axin2^(+/−)doublemutant mice were generated. The size of 3-week-oldAxin1^(Prx1)/Axin2^(+/−)double mutant mouse is much smaller than that of(Axin1^(flox/+))^(Prx1)/Axin2^(+/−)mice (no skeletal phenotype comparedto WT mice) and Axin1^(Prx1) KO mice.

FIG. 10(a) shows Axin1^(Prx1)/Axin2^(+/−)double mutant mice (3-week-old)having more severe defects in fibular development. 23 of Axin1^(Prx1) KOmice (total 52 Axin1^(Prx1) KO mice analyzed) had complete missing offibula and 27 of Axin1^(Prx1) KO mice (52 analyzed) had partial fibulardevelopment (middle panel). In contrast, allAxin1^(Prx1)/Axin2^(+/−)double mutant mice (n=15) had complete missingof fibula (right panel). The Axin1^(Prx1)/Axin2^(+/−)double KO mice havefeatures resembling to proximal femoral focal deficiency (PFFD)phenotypes (right panel).

FIG. 10(b) shows Axin1^(Prx1)/Axin2^(+/−)double KO mice having femoraldefects phenotype resembling that of PFFD, which include: i) femorallength is reduced; ii) the femoral head is not fully developed; iii) noosseous connection between the femoral shaft and head; and iv) dysplasiaof acetabulum.

FIG. 11 shows Axin1^(Prx1)/Axin2^(+/−)double KO mice having severedefects in joint fusion in forelimb. X-ray of forelimbs of 3-week-old(Axin1^(flox/+))^(Prx1)/Axin2^(+/−)(same as Cre-negative control mice),Axin1^(Prx1) KO and Axin1^(Prx1)/Axin2^(+/−)double KO mice wereanalyzed. More severe elbow joint fusion was observed inAxin1^(Prx1)/Axin2^(+/31) mice. Arrows indicate the fusion of humerusand radius in Axin1^(Prx1) KO mice and Axin1^(Prx1)/Axin2^(+/−)double KOmice.

FIG. 12 shows Axin1^(Prx1)/Axin2^(+/31) double KO mice having severedefects in joint fusion in forelimb. μCT analysis of elbow joints wereperformed in 3-week-old Cre⁻, Axin1^(Prx1) KO andAxin1^(Prx1)/Axin2^(+/−)double KO mice. Medial cross sections of elbowjoint through the reconstructed volumes were generated. More severeelbow joint fusion is observed in Axin1^(Prx1)/Axin2^(+/−)double KOmice. The arrow in the central figure indicates the fusion of humerusand radius in Axin1^(Prx1) KO mice. The upper arrow in the right figureindicates the severe fusion of humerus and radius inAxin1^(Prx1)/Axin2^(+/−)double KO mice. The lower arrow in the rightfigure indicates the fusion of humerus and ulna inAxin1^(Prx1)/Axin2^(+/−)double KO mice.

FIG. 13 shows Axin1^(Prx1)/Axin2^(+/−)double KO mice having more severetarsal coalition (TC) phenotypes. μCT analysis was performed in anklejoint of 3-week-old Cre⁻, Axin1^(Prx1) KO andAxin1^(Prx1)/Axin2+/−double KO mice. 3D images showed that most of thetarsal element of ankle joint, 2nd, 3rd, and 4th & 5th tarsal elements,centrale (C) Tibiale (T), intermedium (I) (astragalus) and Fibulare (F)(calcaneum), are fused in Axin1^(Prx1)/Axin2^(+/−)double KO mice(arrow).

FIG. 14 shows Axin1^(Prx1)/Axin2^(+/−)double KO mice having severedefects in fusion of carpal elements in forelimb. μCT analysis of wristjoint of 3-week-old Cre-, Axin1^(Prx1) KO andAxin1^(Prx1)/Axin2^(30 /−)double KO mice showed that more severe wristjoint fusion was observed in Axin1^(Prx1)/Axin2^(30 /−)mice. The 3rddistal carpal, 4th/5th distal carpal, ulnare (U) and ulnar sesamoid (UI)formed a continuous element in the Axin1^(Prx1)/Axin2^(30 /−)double KOmice (arrow).

FIG. 15 shows Axin1^(Prx1)/Axin2^(30 /−)double KO mice having severedefects in joint fusion in forelimb. μCT analysis of forelimb of3-week-old Cre⁻, Axin1^(Prx1) KO and Axin1^(Prx1)/Axin2^(30 /−)double KOmice showed that more severe elbow joint fusion was observedAxin1^(Prx1)/A in2^(30 /−)mice. Lower left arrows indicate the fusion ofhumerus and radius in Axin1^(Prx1) KO mice andAxin1^(Prx1)/Axin2^(30 /−)double KO mice. The right arrow indicates thesevere fusion of humerus and ulna in Axin1^(Prx1)/Axin2^(30 /−)double KOmice. The upper left arrows indicate that the deltoid tuberosity wasalmost completely missing in Axin1^(Prx1) KO mouse and completely absentin Axin1^(Prx1)/Axin2^(30 /−)double KO mice.

FIG. 16 shows that deletion of one allele of β-catenin significantlyreversed joint fusion phenotype of Axin1^(Prx1) KO mice. Xrayradiographic analysis of forelimb of 12-week-old(Axin1^(flox/+)/β-catenin^(flox/+))^(Prx1) (a), Axin1^(Prx1) (b) and(Axin1^(flox/flox)/β-catenin^(flox/+))^(Prx1) (c) mice showed that theelbow joint fusion was reversed in(Axin1^(flox/flox)/β-catenin^(flox/+))^(Prx1) mice (c). The arrow in (b)indicates that humerus and radius are fused in Axin1^(Prx1) KO mice andthe arrow in (c) indicates that the humerus and radius are clearlyseparated in Axin1^(flox/flox)/β-cat^(flox/+))^(Prx1) mice.

FIG. 17(A-C) shows that deletion of one allele of β-cateninsignificantly reversed joint fusion phenotype of Axin1^(Prx1) KO mice.X-ray radiographic analysis of forelimb of 12-week-old(Axin1^(flox/+)/β-catenin^(flox/+))^(Prx1) (a), Axin1^(Prx1) (b) and(Axin1^(flox/flox)/β-catenin^(flox/+))^(Prx1) (c) mice showed that theelbow joint fusion was reversed in(Axin1^(flox/flox)/β-catenin^(flox/+))^(Prx1) mice (c). Arrows in (b)indicate that the humerus and radius are fused in Axin1^(Prx1) KO miceand arrows in (c) indicate the humerus and radius are clearly separatedin (Axin1^(flox/flox)/β-catenin^(flox/+))^(Prx1) mice.

FIG. 18(A-B) shows that defects in fibular development of Axin1^(Prx1)KO mice can be rescued by deletion of one allele of β-catenin. (A) X-rayradiographic analysis showed that defects in fibular developmentobserved in 12-week-old Axin1^(Prx1) mice (middle panel) weresignificantly reversed by deletion of one allele of β-catenin (lowerpanel). Dark arrows show bone mass increase. (B) μCT analysis showedthat bone volume (BV) was increased to 92% in Axin1^(Prx1) KO mice.Deletion of one allele of β-catenin caused BV reduction from 92 to 71%.

FIG. 19 Shows that deletion of one allele of β-catenin significantlyreversed fibular defects observed in Axin1^(Prx1) KO mice. Axin1^(Prx1)KO mice were bred with β-catenin^(flox/+)mice to produce(Axin1^(flox/flox)/β-catenin^(flox/+))^(Prx1) double mutant mice.Axin1^(Prx1) mice (12-week-old) showed fibular defects (middle panel)and these were reversed by deletion of one allele of β-catenin (rightpanel).

FIG. 20(A-G) shows that BMP signaling is up-regulated in Axin1^(Prx1)conditional KO mice. Bmp2 (A), Bmp4 (A), Bmp7 (C), Gdf5 (D), Gremlin(E), Msx1 (F) and Msx2 (G). Total mRNA was extracted from E14.5 limbs ofCre-negative and Axin1^(Prx1) KO embryos. Realtime PCR analysis showedthat expression of Bmp2 (A), Bmp4 (B), Gdf5 (D), Gremlin (E), and Msx2(G) was significantly up-regulated Axin1^(Prx1) embryos.

FIG. 21 show that inhibition of BMP signaling reversed joint fusionphenotype of Axin1^(Prx1) KO mice. BMP inhibitor dorsomorphin (2.5mg/kg, i.p. injection, single dose) was injected into pregnant mothersat E9.5 stage. Histological analysis showed that the humerus, radius andulna are clearly separated in Axin1^(Prx1) KO mice (right panel, arrow)which were treated with dorsomorphin. In contrast, histological sectionsof elbow joint of E18.5 Axin1^(Prx1) KO mice without treatment of BMPinhibitor showed that the humerus and radius was fused in Axin1^(Prx1)KO embryos (left panel, arrow). Lower panels: higher magnification.

FIG. 22 shows that inhibition of BMP signaling reversed fibular defectsobserved in Axin1^(Prx1) KO mice. BMP inhibitor dorsomorphin was givento pregnant mothers at E9.5 stage (Axin1^(Prx1) mice were bred withAxin1^(flox/flox) mice) (2.5 mg/kg, i.p. injection, single dose).Histology (Alcian Blue/Hematoxylin & Orange G staining) of hind limb ofE18.5 Cre⁻and Axin1^(Prx1) KO mice showed that administration of BMPinhibitor to Axin1^(Prx1) KO mice significantly reversed fibular defectsobserved in Axin1^(Prx1) KO mice (right panel).

FIG. 23 shows inhibition of BMP signaling reversed fibular defectsobserved in Axin1^(Prx1) KO mice. BMP inhibitor, dorsomorphin, was givento pregnant mothers at E13.5 stage (Axin1^(Prx1) mice were bred withAxin1^(flox/flox) mice) (5 mg/kg, i.p. injection, single dose).Administration of BMP inhibitor to Axin1^(Prx1) KO mice almostcompletely reversed fibular defects observed in Axin1^(Prx1) KO mice(right panel).

FIG. 24 shows inhibition of BMP signaling reversed elbow joint fusionphenotype in Axin1^(Prx1) KO mice. BMP inhibitor, dorsomorphin, wasgiven to pregnant mothers at E9.5 stage (2.5 mg/kg, i.p. injection,single dose). Micro-CT analysis of elbow joint of 6-week-old mice showedthat humerus (H), radius (R) and ulna (U) are clearly separated inAxin1^(Prx1) KO mice (right panel, red arrow) treated with dorsomorphin.No significant difference in the elbow joint was observed in Crenegative Axin1^(flox/flox) mice treated with dorsomorphin compared towild type mice.

FIG. 25 shows inhibition of Wnt signaling reversed joint fusionphenotype of Axin1^(Prx1) KO mice. Wnt inhibitor, iCRT14 (2.5 mg/kg,i.p. injection, single dose) was injected into pregnant mothers at E9.5stage. Samples were collected at E18.5. Histological analysis showedthat humerus (H), radius (R) and ulna (U) are clearly separated inAxin1^(Prx1) mice (right panel, red arrow) treated with iCRT14. Incontrast, histological sections of the elbow joint of E18.5 Axin1^(Prx1)KO mice without treatment of Wnt inhibitor showed that humerus(H) andradius (R) were fused (left panel, black arrow). iCRT14 did not affectelbow joint formation in the Cre negative embryos (middle panel). Lowerpanels show higher magnification.

FIG. 26 shows inhibition of Wnt signaling reversed fibular defectobserved in Axin1^(Prx1) KO mice. Wnt inhibitor, iCRT14 (2.5 mg/kg, i.p.injection, single dose) was injected into pregnant mothers at E9.5stage. Samples were collected at E18.5. Alcian Blue/Hematoxylin & OrangeG staining of the hind limb of E18.5 Cre negative and Axin1^(Prx1) KOmice showed that administration of Wnt inhibitor to Axin1^(Prx1) micesignificantly reversed fibular defect (right panel, green arrow)observed in Axin1^(Prx1) KO mice.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Definitions

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. In case of conflict, thepresent document, including definitions, will control. Preferred methodsand materials are described below, although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention.

The uses of the terms “a” and “an” and “the” and similar references inthe context of describing the invention (especially in the context ofthe following claims) are to be construed to cover both the singular andthe plural, unless otherwise indicated herein or clearly contradicted bycontext. Recitation of ranges of values herein are merely intended toserve as a shorthand method of referring individually to each separatevalue falling within the range, unless otherwise indicated herein, andeach separate value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”, “for example”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

The term “therapeutic effect” as used herein means an effect whichinduces, ameliorates or otherwise causes an improvement in thepathological symptoms, disease progression or physiological conditionsassociated with or resistance to succumbing to a disorder, for example agenetic bone disease, of a human or veterinary subject. The term“therapeutically effective amount” as used with respect to a drug meansan amount of the drug which imparts a therapeutic effect to the human orveterinary subject. The therapeutically effective amount may bedelivered to the subject in-utero (before birth) and after the birth ofthe subject.

Methods for Treating a Genetic Bone Disease

For the purpose of promoting an understanding of the principles of theinvention, reference will now be made to embodiments, some of which areillustrated in the drawings, and specific language will be used todescribe the same. It will nevertheless be understood that no limitationof the scope of the invention is thereby intended. Any alterations andfurther modifications in the described embodiments, and any furtherapplications of the principles of the invention as described herein arecontemplated as would normally occur to one skilled in the art to whichthe invention relates. In the discussions that follow, a number ofpotential features or selections of methods, methods of analysis, orother aspects, are disclosed. It is to be understood that each suchdisclosed feature or features can be combined with the generalizedfeatures discussed, to form a disclosed embodiment of the presentinvention.

Axin1 is a critical negative regulator of the canonical Wnt-signalingpathway. It is a concentration-limiting factor in the β-catenindegradation complex. Axin1 null mutant mouse embryos typically die atembryonic day 9.5, precluding direct genetic analysis of the roles ofAxin1 in many developmental and physiological processes using thesemutant mice. Mice may be generated carrying two directly repeated IoxPsites flanking the exon 2 region of the Axin1 gene. Suchfloxed-allele-carrying mice (Axin1^(flox/flox)) mice appear normal andfertile. Upon crossing the Axin1^(flox/flox) mice to the CMV-Cretransgenic mice to generate Axin1^(Prx1) mice, the IoxP-flanked exon 2region that encodes the N-terminus and the conserved regulation ofG-protein signaling domain is efficiently deleted by Cre-mediatedexcision in vivo. Mouse embryos homozygous for the Cre/loxP-mediateddeletion of exon 2 of the Axin1 gene display embryonic lethality anddevelopmental defects similar to those reported for Axin1^(−/−)mice.This Axin1^(flox/flox) mouse model provides a valuable for systematictissue-specific dissection of the roles of Axin1 in embryonic andpostnatal development and diseases.

The present inventors have found that deletion of Axin1 in limbmesenchymal progenitor cells, through generating Axin1^(Prx1)conditional knockout mice and Axin1^(Prx1)/Axin2^(+/−)double KO mice,led to phenotypes assembling to specific genetic orthopaedic diseases,for example, fibular hemimelia (FH), proximal femoral focal deficiency(PFFD), tarsal coalition (TC) or humeroradial synostosis (HRS). Theyhave found that Beta-catenin signaling is activated in Axin1^(Prx1)conditional knockout mice and that deletion of one allele of β-cateningene reverses disease phenotypes. Furthermore, inhibition of Wntsignaling using chemical inhibitors significantly reversed diseasephenotypes of Axin1^(Prx1) conditional KO mice. This provides evidencethat inhibition of β-catenin is a potential treatment for these geneticorthopaedic diseases.

In addition, bone morphogenetic protein (“BMP”) signaling isup-regulated in Axin1^(Prx1) conditional knockout mice. Inhibition ofBMP signaling using chemical inhibitor of BMP signaling also reverseddisease phenotypes of Axin1^(Prx1) conditional knockout mice,demonstrating that this specific BMP signaling inhibitor may be used totreat the diseases of such genetic bone diseases.

One aspect of the present invention provides a method for treating asubject having a genetic bone disease. In one embodiment, the methodincludes administering a composition including a therapeuticallyeffective amount of an inhibitor of beta-catenin signaling. The diseasemay be, for example, fibular hemimelia (FH), proximal femoral focaldeficiency (PFFD), tarsal coalition (TC) or humeroradial synostosis(HRS).

The composition may be administered in-utero and/or after birth of thesubject. For example, when the subject is a human fetus, the compositionmay be administered in the first, second or third trimester ofdevelopment. The subject may be a subject having an elevated level ofβ-catenin signaling. In certain embodiments, the level of β-cateninsignaling is determined and the treatment administered on the basis ofthis level.

In certain embodiments, the inhibitor of beta-catenin signaling isLGK-974 ([2,4′-Bipyridine]-5-acetamide,2′,3-dimethyl-N-[5-(2-pyrazinyl)-2-pyridinyl]-), IWP-2 (Acetamide,N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio],iCRT3(2-[[[2-(4-ethylphenyl)-5-methyl-4-oxazolyl]methyl]thio]-N-(2-phenylethyl)acetamide),iCRT 14(:5-[[2,5-Dimethyl-1-(3-pyridinyl)-1H-pyrrol-3-yl]methylene]-3-phenyl-2,4-thiazolidinedion),ICG 001((6S,9aS)-Hexahydro-6-[(4-hydroxyphenyl)methyl]-8-(1-naphthalenylmethyl)-4,7-dioxo-N-(phenylmethyl)-2H-pyrazino[1,2-a]pyrimidine-1(6H)carboxamide),XAV-939(2-(4-(trifluoromethyl)phenyl)-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-4-ol),KY02111(Benzenepropanamide,N-(6-chloro-2-benzothiazolyl)-3,4-dimethoxy-), orphysiologically-acceptable salts thereof. In certain embodiments, acombination or at least two of these compounds is administered.

Another aspect of the invention provides a method for treating theorthopedic disease including administering a composition including atherapeutically effective amount of an inhibitor of bone morphogeneticprotein (BMP) signaling. The composition may be administered asdescribed above, for example in-utero and/or after birth of the subject.The subject may be a subject having an elevated level of BMP signaling.In certain embodiments, the level of BMP signaling is determined and thetreatment administered on the basis of this level.

In certain embodiments, the inhibitor of BMP signaling is dorsomorphin(6-[4-[2-(1-Piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine),K 02288 (3-[(6-Amino-5-(3,4,5-trimethoxyphenyl)-3-pyridinyl]phenol),DMH-1(4-[6-[4-(1-Methylethoxy)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline),LDN 193189(4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinolone)or physiologically-acceptable salts thereof. For example, the inhibitorof BMP signaling is dorsomorphin dihydrochloride or LDN 193189trihydrochloride. In certain embodiments, a combination or at least twoof these compounds is administered. In other embodiments, a combinationof at least one inhibitor of beta-catenin signaling and at least oneinhibitor of BMP signaling is administered.

Pharmaceutical Compositions

Another aspect of the present invention provides pharmaceuticalcompositions including at least one inhibitor of beta-catenin signalingor at least one inhibitor of BMP signaling or a combination thereof.

The pharmaceutical compositions can be in the form of, for example,tablets, pills, dragees, hard and soft gel capsules, granules, pellets,aqueous, lipid, oily or other solutions, emulsions such as oil-in-wateremulsions, liposomes, aqueous or oily suspensions, syrups, alixiers,solid emulsions, solid dispersions or dispersible powders. Inpharmaceutical compositions for oral administration, the agent may beadmixed with commonly known and used adjuvants and excipients, forexample, gum arabic, talcum, starch, sugars (such as, e.g., mannitose,methyl cellulose, lactose), gelatin, surface-active agents, magnesiumstearate, aqueous or non-aqueous solvents, paraffin derivatives,cross-linking agents, dispersants, emulsifiers, lubricants, conservingagents, flavoring agents (e.g., ethereal oils), solubility enhancers(e.g., benzyl benzoate or benzyl alcohol) or bioavailability enhancers(e.g. GELUCIRE). In the pharmaceutical composition, the agent may alsobe dispersed in a microparticle, e.g. a nanoparticulate, composition.

For parenteral administration, the agent or pharmaceutical compositionsof the agent can be dissolved or suspended in a physiologicallyacceptable diluent, such as, e.g., water, buffer, oils with or withoutsolubilizers, surface-active agents, dispersants or emulsifiers. As oilsfor example and without limitation, olive oil, peanut oil, cottonseedoil, soybean oil, castor oil and sesame oil may be used. More generally,for parenteral administration the agent or pharmaceutical compositionsof the agent can be in the form of an aqueous, lipid, oily or other kindof solution or suspension or even administered in the form of liposomesor nano-suspensions.

Mode of Administration

The pharmaceutical compositions may be administered by any method thatallows for the delivery of a therapeutic effective amount of the agentto the subject. Modes of administration can include, but are not limitedto oral, topical, transdermal and parenteral routes, as well as directinjection into a tissue and delivery by a catheter. Parenteral routescan include, but are not limited to subcutaneous, intradermal,intra-articular, intravenous, intraperitoneal and intramuscular routes.In one embodiment, the route of administration is by topical ortransdermal administration, such as by a lotion, cream, a patch, aninjection, an implanted device, a draft or other controlled releasecarrier. Routes of administration include any route which directlydelivers the composition to the systemic circulation (e.g., byinjection), including any parenteral route.

When the composition is delivered in-utero, it may be delivered by forexample, a parenteral route, for example, by intraperitoneal delivery.

One embodiment of the method of the invention includes administering thecomposition in a dose, concentration and for a time sufficient toprevent the development of, or to lessen the extent of the genetic bonedisease. Certain embodiments include administering systemically thecomposition in a dose between about 0.1 micrograms and about 100milligrams per kilogram body weight of the subject, between about 0.1micrograms and about 10 milligrams per kilogram body weight of thesubject, between about 0.1 micrograms and about 1 milligram per kilogrambody weight of the subject. In practicing this method, the compositioncan be administered in a single daily dose or in multiple doses per day.This treatment method may require administration over extended periodsof time. The amount per administered dose or the total amountadministered will be determined by the physician and will depend on suchfactors as the mass of the patient, the age and general health of thepatient and the tolerance of the patient to the compound.

Methods for Identifying Compounds for Treating a Genetic OrthopedicsDisease

Another aspect of the invention provides methods for identifyingcompounds for treating a genetic orthopedic disease. In one embodiment,the method includes administering a test compound to an Axin1^(Prx1)non-human mammalian embryo and examining an elbow joint of the embryo todetermine the extent of separation of the humerus, radius and ulna. Inone embodiment, the non-human mammalian embryo is a mouse embryo.

compounds that exhibit at least a predetermined separation of thehumerus, radius and ulna are identified as compounds for treating thegenetic orthopedic disease. In one embodiment, the examination is byx-ray analysis. In some embodiments, the method includes comparing theextent of separation of the humerus, radius and ulna to that observed ina Axin1^(Prx1) non-human mammalian embryo, for example a Axin1^(Prx1)mouse embryo, that has not been exposed to the test compound.

Embodiments of the invention will be further described in the followingexamples, which do not limit the scope of the invention described in theclaims.

Example 1—Generation of a Conditional Allele of Mouse Axin1

To circumvent the early lethality (E9.5) observed in Axin1 globalknockout (KO) mice (Zeng et al., 1997) and to assess the specific roleof Axin1 in skeletal development and postnatal bone homeostasis, wegenerated a strain of mice that carry targeted alleles of Axin1(Axin1^(flox/flox) mice) allowing for conditional deletion of the Axin1gene when bred with mice expressing Cre recombinase (Xie et al., 2011).

The function of Axin1 in vivo is poorly understood because Axin1 KO miceare embryonic lethal at E9.5 or E10.5 (Zeng et al., 1997). We generateda conditional targeted allele of Axin1, the Axin1^(flox/flox), mice. Theshort (2.6-kb) and long (7.4-kb) homologous arms for the targetingvector were isolated from a 129/sv mouse BAC library (RPCI-22 422B5).Since the exon2 of the Axin1 gene has been demonstrated to be essentialfor the function of Axin1 (Zeng et al., 1997), exon2 was flanked by twoIoxP sites in the targeting construct. A neomycin (neo) cDNA cassette,under the control of thymidine kinase (tk) promoter and flanked by twoFRT sites was introduced at the 3′-end of the second IoxP site. Thediphtheria toxin A-fragment (DTA) driven by the phosphoglycerate kinase(PGK) promoter was also inserted at the 5′- and 3′-end of the vector,respectively (Xie et al., 2011). The linearized targeting construct wasthen introduced into ES cells by electroporation. ES clones resistant toG418 were screened for homologous recombination by Southern blotting.Two positive clones were injected into C57BL/6J blastocysts at thetransgenic facility of the University of Texas (Houston, Tex.). Threechimeric mice were obtained, and one of these transmitted the targetedAxin1 locus to subsequent generations. The mice were genotyped bySouthern blotting by 5′ probe and by PCR using DNAs extracted from tailtissues (Xie et al., 2011). The neomycin cassette was deleted bycrossing the resulting mice to the Rosa26-FLP mice (Farley et al., 2000;Xie et al., 2011). The Axin^(flox/flox) mice were viable and fertile,and did not present any recognizable phenotype.

The Axin1^(−/−)homozygous embryos died at E9.5 or E10.5, displaying awide spectrum of abnormalities including incomplete closure ormalformation of head folds, crooked neural tube, cardia bifida andduplication of embryonic axis (Perry et al., 1995; Zeng et al., 1997).The Axin1^(del/del) mice (CMV-Cre;Axin1^(flox/flox)) exhibit recessiveembryonic defects similar to those caused by the null allele ofAxin1^(Tg1) (Perry et al., 1995; Zeng et al., 1997; Chia et al., 2009).Twenty-seven embryos from intercrossed Axin1^(del/+)mice were examinedat E10.5 and seven Axin1^(del/del) homozygotes were found, consistentwith the expected Mendelian ratio. Eighteen embryos from intercrossedAxin1^(del/+)mice were also examined at E9.5. Four Axin1^(del/del)homozygotes were found as the expected frequency. Next we performedWestern blot analysis using cell lysates extracted from whole embryos(Xie et al., 2011). Strong Axin1 band was observed in theAxin1^(del/+)cells but no signal was visible for Axin1^(del/del) cells.All embryos of the homozygotes were severely abnormal. They weresignificantly smaller than their wild-type or heterozygous littermatesand displayed underdeveloped head folds and open head folds (Xie et al.,2011). The embryos of heterozygotes were indistinguishable from that ofwild-type littermates (data not shown). Therefore, the deleted alleleshould represent a null allele of Axin1.

Example 2—Specific Deletion of Axin1 in Limb Mesenchymal Stem Cells(MSCs) Leads to Fibular Hemimelia (FH), Tarsal Coalition (TC) andHumeroradial Synostosis (HRS) Phenotypes

Inactivation of Axin1 in limb MSCs results in defects in fibuladevelopment. In order to determine the role of Axin1 in skeletogenesis,we generated Axin1^(Prx1) mice by breeding the Axin1^(flox/flox) micewith Prx1-Cre transgenic mice (Logan et al., 2002) in which the Creexpression is under the control of the Prx1 promoter. This Prx1(paired-related homeobox gene-1) regulatory element leads to Creexpression throughout the early limb bud mesenchyme and in a subset ofcraniofacial mesenchyme. Crossing Prx1-Cre mice to a reporter mouseharboring a floxed Cre-reporter cassette revealed that recombinaseactivity is first observed in the earliest limb bud at E9.5. By early tomid-bud stages at E10.5 recombination is essentially complete in allmesenchymal cells in the limb (Logan et al., 2002). Prx1-Cre mice weregenerated in Dr. Cliff Tabin's lab (Harvard University) and weredeposited in Jackson Lab. We obtained this mouse strain from Jackson Lab(Bar Harbor, Me., C57BL/6 background).

We first examined skeletal development at E13.5, E16.5 and postnatal day7 stages by Alizarin red/Alcian blue staining. The one not notabledefect is the presence of various fibular deficiencies in theAxin1^(Prx1) homozygous embryos and postnatal mice (FIG. 1).Radiographic analysis of 1- and 2-week-old mice showed that the absenceof the fibula in some of Axin1^(Prx1) mice was complete or almostcomplete (>50%, 27/52) where only a distal, vestigial fragment waspresent (FIG. 2A). The other Axin1^(Prx1) mice had partial absence ofthe fibula (30-50%, 23/52) in which the proximal portion of the fibulawas absent while the distal portion was present but could not supportthe ankle (FIG. 2A). And the mild fibular hypoplasia was observed insome Axin1^(Prx1) mice (2/52), in which the fibula was absent less than30% of its length (FIG. 2B, 2^(nd)left and far right). These resultsindicate that Axin1 plays an essential role in fibular development. Inaddition to the fibular absence or hypoplasia, we also observed severaladditional skeletal defects in Axin1^(Prx1) mice. All femora ofAxin1^(Prx1) mice were shorter and wider than those of theirCre-negative littermates (FIG. 2B, far right). The bowed tibia wasobserved (FIG. 2B, 2^(nd) left). The valgus ankle was also seen and thetarsal coalitions were also found in the Axin1^(Prx1) mice. Histologicalanalysis showed that partially developed fibular tissues dissected fromAxin1^(Prx1) mice had high bone mass and poorly developed growth plate(FIG. 2C). The number of chondrocytes was significantly reduced and thestructure of growth plate chondrocytes was disorganized (FIG. 2C). It isinteresting to note that these skeletal defects observed in Axin1^(Prx1)mice have been suggested to be the key features of FH disease in humans(Thompson et al., 1957; Achterman and Kalamchi, 1979; Stanitski andStanitski, 2003). In summary, our preliminary data suggest that Axin1 isrequired for fibular development.

To determine the role of Axin1 in other cell populations, we bredAxin1^(flox/flox) mice with Osx1-Cre (Rodda and McMahon, 2006) orCol2-Cre (Ovchinnikov et al., 2000) transgenic mice and generatedAxin1^(Osx1) and Axin1^(Col2) conditional KO mice. We didn't observefibular defects in Axin1^(Osx1) (targeting deletion of Axin1 inOsx1-expressing osteoblast precursor cells) or Axin1^(Col2) mice(targeting deletion of Axin1 in Col2-expressing chondrocytes) (FIG. 3).These results suggest that Axin1 plays a specific role in controllingfibular development within the MSC population.

Inactivation of Axin1 in limb MSCs resulted in synovial joint fusion inmice. Another notable defect observed in Axin1^(Prx1) mice was that thesynovial joints were fused. Specifically, the fusion of joint wasapparent at elbow, where the humeroradial and humerolunar fusions wereobserved. Alizarin red/Alcian blue stained skeletal preparations offorelimbs showed that Axin1^(Prx1) embryos exhibited single jointfusions of the humerus and radius, humerus and ulna in the forelimbs ofE13.5 and E16.5 embryos and postnatal P7 mice, whereas control embryosshowed a clear cavitation of joint and separation of the three skeletalelements (FIG. 4). Analysis by radiographs in P12 Axin1^(Prx1) mice(FIG. 5A) and by μCT in P30 Axin1^(Prx1) mice (FIG. 5B) revealed thatthe humeroradial and humerolunar fusions in the forelimbs ofAxin1^(Prx1) mice. Histologic examination of Axin1^(Prx1) forelimbsections at P4 and P15 showed humeroradial fusions (FIG. 6). Inaddition, radiographic and μCT analyses revealed fusion of tarsalelements in Axin1^(Prx1) mice. The 2^(nd) distal tarsal, 3^(rd) distaltarsal, 4^(th) & 5^(th) distal tarsal and centrale form a continuousskeletal element (FIG. 7). Analysis by μCT also exhibited defect of thedeltoid tuberosity in 3- and 6-week-old Axin1^(Prx1) mice (FIG. 8).Taken together, Axin1 is required for synovial joint formation.

Example 3—Axin1^(Prx1)/Axin2^(+/−)Double Mutant Mice Have Much SevereDefects in Fibular Hemimelia (FH), Proximal Femoral Focal Deficiency(PFFD), Tarsal Coalition (TC) and Humeroradial Synostosis (HRS)Phenotypes than Axin1^(Prx1) Mice

Axin2 is the homolog of Axin1 and is 44% identical to Axin1 in theiramino acid sequences. It has been reported that Axin1 and Axin2 proteinsare functionally equivalent (Chia and Costantini, 2005). Axin2 null mice(Axin2^(−/−)) appeared craniofacial defects (Yu et al., 2005) and ourlab has previously demonstrated the high bone mass phenotype in adultAxin2^(−/−)mice (Yan et al., 2009). Although Axin2 mutant mice have noapparent fibula and synovial joint phenotype, we hypothesized thatdeletion of Axin1 in combination with deletion of one allele of Axin2gene might result in more severe consequences for fibular developmentand synovial joint formation. The body size of the 3 week oldAxin1^(Prx1)/Axin2^(+/−)double mutant mice is much smaller thanAxin1^(Prx1) mice (FIG. 9). To date, we have analyzed 52 Axin1Prx1 miceand all of them have a fibular deficiency phenotype, indicating thatAxin1 is required for fibular development. 27/52 had almost completemissing of fibula (>50% loss), 23/52 had partial fibular development(30-50% loss), and 2/52 had weak fibular defects (<30% loss). Incontrast, all Axin1^(Prx1)/Axin2^(+/−)double mutant mice (n=15)displayed a complete absence of the fibula (FIG. 10(a)). These resultssuggest that both Axin1 and Axin2 are essential for fibularmorphogenesis and development.

In addition, the Axin1^(Prx1)/Axin2^(+/−)double KO mice have featuresresembling to proximal femoral focal deficiency (PFFD) phenotypes (FIG.10(a), right panel) Axin1^(Prx1)/Axin2^(+/−)double KO mice have femoraldefects phenotype resembling that of PFFD, which include: i) femorallength is reduced; ii) the femoral head is not fully developed; iii) noosseous connection between the femoral shaft and head; and iv) dysplasiaof acetabulum (FIG. 10(a), 10(b)).

As expected, the Axin1^(Prx1)/Axin2^(+/−)mice showed more severe jointfusion in the elbow, wrist and ankle joint.Axin1^(Prx1)/Axin2^(+/−)double mutant mice exhibit more severehumeroradial and humerolunar fusions (FIGS. 11 and 12). Analysis byradiographs and μCT revealed that almost all tarsal elements (2^(nd)distal tarsal, 3^(rd) distal tarsal, 4^(th) & 5^(th) distal tarsal,centrale, tibiale, intermedium and fibulare) were fused (FIG. 13) in3-week-old Axin1^(Prx1)/Axin2^(+/−)double mutant mice. The joint ofwrist of the Axin1^(Prx1)/Axin2^(+/−)mice also showed more severephenotypes, the 3^(rd) distal carpal, 4^(th) & 5^(th) distal carpal,ulnare and ulnar sesamoid formed a continuous element (FIG. 14). Thedeltoid tuberosity was totally absent in theAxin1^(Prx1)/Axin2^(+/−)double mutant mice (FIG. 15). These data revealthat both Axin1 and Axin2 play essential roles in synovial jointdevelopment and indicate that endogenous Axin2 can partially compensatefor the absence of Axin1 during joint formation.

Example 4—Deletion of One Allele of β-Catenin Significantly ReversedFibular Hemimelia (FH) and Humeroradial Synostosis (HRS) Phenotypes inAxin1^(Prx1) Mice

Since Axin1 is a negative regulator of canonical Wnt signaling pathway,deletion of Axin1 will elevate β-catenin protein levels. If defects inskeletal development in Axin1^(Prx1) mice are due to elevated levels ofβ-catenin, reducing the β-catenin (Ctnnb1) gene dosage might fully orpartially correct defects observed in Axin1^(Prx1) mice. On the otherhand, if defects of skeletal development are β-catenin-independent,reducing the β-catenin gene dosage should have no effect. To test thishypothesis, we looked at genetic interaction between Axin1 and β-cateninin synovial joint formation. We found that defect in elbow joint inAxin1^(Prx1) mice were alleviated in(Axin1^(flox/flox)/β-catenin^(flox/+))^(Prx1) mice (FIGS. 16 and 17).The radius and humerus are clearly separated in(Axin1^(Prx1)/β-catenin^(flox/+))^(Prx1) mice, although the joint isstill dislocated (FIGS. 16 and 17). We found that deletion of one alleleof the β-catenin gene in Axin1^(Prx1) mice caused reduction of BV from92 to 71% (FIG. 18A) and significantly reversed defects in fibulardevelopment (FIG. 18B). However, the rescuing is not complete and thefibula did not extend to the proximal end of tibia as observed in WTmice (FIG. 18B, FIG. 19). The bone density of the tibia is much higherand the shape of tibia is much wider in double mutant mice thanCre-negative mice (FIG. 18B, FIG. 19). These results demonstrate thatAxin1 regulates the joint formation, fibular development and bone volumeat least partially through the canonical Wnt/β-catenin pathway.

Example 5—Inhibition of BMP Signaling Significantly Reversed FH and HRSPhenotypes Observed in Axin1^(Prx1) Mice

In previous studies, we found that Bmp2 and Bmp4 expression wasup-regulated in Axin2 KO mice (Yen et al., 2009). To determine if BMPsignaling is up-regulated in Axin1^(Prx1) mice, we extracted total RNAfrom E14.5 hind limb of Cre-negative and Axin1^(Prx1) embryos. Weanalyzed expression of several Bmp genes and BMP target genes and foundthat expression of Bmp2, Bmp4, Gdf5, Gremlin and Msx2 was significantlyup-regulated in the limb tissues of Axin1^(Prx1) embryos (FIG. 20).

Because it is well documented that BMP signaling acts on inhibition ofsynovial joint formation (Brunet et al., 1998, Tsumaki et al., 2002; Zouet al., 1997) and the Axin1^(Prx1) mice and Axin1^(Prx1)/Axin2^(+/−)miceshowed multiple joint fusions involving humeroradial, carpal and tarsaljoint that are very similar to those found noggin mutant mice, it islikely that BMP signaling pathway is one of those immediately downstreampathway of Axin1/β-catenin signaling during skeletal development. Totest this possibility, we investigated if inhibition of BMP signalingwill reverse skeletal defects of the Axin1^(Prx1) mice. We injectedAxin1^(Prx1) mice intraperitoneally with BMP pathway inhibitor,dorsomorphin (2.5 mg/kg), which has recently been shown to inhibitBMPR-IA(ALK3), BMPR-IB (ALK6) and ALK2 activity (Yu et al., 2008), orvehicle at E9.5 stage. The embryos were collected at E18.5. Alcianblue/H&E staining on Axin1^(Prx1) elbow joint sections at E18.5 revealedthat the humerus, radius and ulna were clearly separated in Axin1^(Prx1)KO treated with treatment of BMP inhibitor (FIG. 21). Dorsomorphin didnot affect joint formation in the Cre negative embryos. These resultsdemonstrated that the humeroradial and humeroulnar fusions inAxin1^(Prx1) mice were rescued by blocking BMP signaling. Histologicexamination of hindlimb sections of E18.5 embryos showed that fibulardefects in Axin1^(Prx1) embryos were significantly rescued by thetreatment of dosomorphin (FIG. 22). In addition, BMP inhibitordorsomorphin (2.5 mg/kg, i.p. injection, single dose) was injected intopregnant mothers at E9.5 stage. Micro-CT analysis of elbow joint of6-week-old mice showed that the humerus (H), radius (R) and ulna (U) areclearly separated (FIG. 24, right panel, red arrow) in Axin1^(Prx1) KOmice which were treated with dorsomorphin. And the fibular defect inAxin1^(Prx1) mice was significantly also rescued by dosomorphintreatment (data not shown).

However, when we injected Axin1^(Prx1) embryos with dorsomorphin (5mg/kg) at E13.5 stage, the result of μCT analysis at 6 weeks of ageshowed that fibular defects in Axin1^(Prx1) mice was significantlyrescued by dosomorphin treatment (FIG. 23), but the elbow and tarsaljoint fusions were still observed in the in Axin1^(Prx1) mice (data notshown), suggesting that the timing for BMP inhibitor administration isvery important. Taken together, these results indicate that BMPsignaling pathway is a central downstream effector of Axin1/β-cateninsignaling during skeletal development and the use of BMP inhibitor mayrepresent a potential therapy for FH and HRS synostosis diseases.

Example 6—Inhibition of Wnt Signaling Significantly Reversed FH and HRSPhenotypes Observed in Axin1^(Prx1) Mice

Our preliminary data demonstrated that deletion of one allele ofβ-catenin in Axin1^(Prx1) mice significantly reversed FH and HRSphenotypes observed in in Axin1^(Prx1) mice, suggesting that FH and HRSphenotypes observed in in Axin1^(Prx1) mice are mediated by elevatedWnt/β-catenin level. To further determine the role of Wnt/β-catenin inskeletal development and to explore if Wnt inhibition could be used as apotential therapeutic treatment for FH/TC/HRS, Axin1^(Prx1) mice(pregnant mother at E9.5 stage) were treated with the Wnt/β-catenininhibitor, iCRT14 (inhibitor of β-catenin responsive transcription) (2.5mg/kg, i.p. injection, single dose). iCRT14 is a small-moleculeinhibitor of nuclear β-catenin function and has been shown tospecifically inhibit Wnt/β-catenin-induced transcription by disruptingthe interaction between β-catenin and TCF4 (Gonsalves et al. 2011). Theembryos were collected at E18.5 stage. Histological analysis showed thatthe HRS phenotype in Axin1^(Prx1) embryos was reversed. The humerus (H),radius (R) and ulna (U) are clearly separated (FIG. 25) in Axin1^(Prx1)mice treated with iCRT14.

A histological examination of hind limb section of E18.5 embryos showedthat the fibular defects Axin1^(Prx1) in mice were almost completelyrescued by iCRT14 treatment (FIG. 26). iCRT14 did not affect jointformation and fibular development in the Cre negative embryos. Theseresults demonstrate that β-catenin is a major signaling event downstreamof canonical Wnt signaling, if not the only one. The results indicatethat Wnt/β-catenin inhibitor represents a potential therapy for FH andHRS.

Although the invention has been described and illustrated with referenceto specific illustrative embodiments thereof, it is not intended thatthe invention be limited to those illustrative embodiments. Thoseskilled in the art will recognize that variations and modifications canbe made without departing from the true scope and spirit of theinvention as defined by the claims that follow. It is therefore intendedto include within the invention all such variations and modifications asfall within the scope of the appended claims and equivalents thereof.

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We claim:
 1. A method for treating an orthopedic disease, comprisingadministering to a subject in need of such treatment a compositioncomprising a therapeutically effective amount of an inhibitor ofbeta-catenin signaling.
 2. The method of claim 1, wherein the disease isselected from the group consisting of fibular hemimelia, proximalfemoral focal deficiency, tarsal coalition and humeroradial synostosis.3. The method of claim 1, wherein the composition is administeredin-utero.
 4. The method of clam 3, wherein the composition isadministered in the third trimester of pregnancy.
 5. The method of claim1, wherein the composition is administered after birth of the subject.6. The method of claim 1, wherein the subject is a subject having anelevated level of beta-catenin signaling.
 7. The method of claim 1,wherein the inhibitor of beta-catenin signaling is selected from thegroup consisting of LGK-974, IWP-2, iCRT 3, iCRT 14, ICG 001, XAV-939,KY02111, and a combination of at least two thereof.
 8. The method ofclaim 7, wherein the inhibitor of β-catenin signaling is iCRT
 14. 9. Themethod of claim 7, wherein the inhibitor β-catenin signaling is XAV-939.10. A method for treating an orthopedic disease, comprisingadministering to a subject in need of such treatment a compositioncomprising a therapeutically effective amount of an inhibitor of bonemorphogenetic protein (BMP) signaling.
 11. The method of claim 10,wherein the disease is selected from the group consisting of fibularhemimelia, proximal femoral focal deficiency, tarsal coalition orhumeroradial synostosis.
 12. The method of claim 10, Wherein thecomposition is administered in-utero.
 13. The method of claim 12,wherein the composition is administered in the third trimester ofpregnancy.
 14. The method of claim 10, wherein the composition isadministered after birth of the subject.
 15. The method of claim 10,wherein the subject is a subject having an elevated level of BMPsignaling.
 16. The method of claim 10, wherein the inhibitor of BMPsignaling is selected from the group consisting of dorsomorphindihydrochloride, K 02288, DMH-1 and LDN 193189 trihydrochloride.
 17. Themethod of claim 16, wherein the inhibitor of BMP signaling isdorsomorphin dihydrochloride.
 18. A method for identifying compounds fortreating a genetic orthopedic disease, comprising: administering a testcompound to a Axin1^(Prx1) mouse embryo; examining a elbow joint of theembryo to determine the extent of separation of humerus, radius andulna, wherein compounds that exhibit at least a predetermined separationof humerus, radius and ulna are identified as compounds for treating thegenetic orthopedic disease.
 19. The method of claim 18, wherein thedisease is selected from the group consisting of fibular hemimelia,proximal femoral focal deficiency, tarsal coalition or humeroradialsynostosis.
 20. The method of claim 18, wherein the examining isperformed by x-ray analysis.